Screening and isolation of cyclooxygenase‐2 inhibitors from Trifolium pratense L. via ultrafiltration,enzyme‐immobilized magnetic beads,semi‐preparative high‐performance liquid chromatography and high‐speed counter‐current chromatography

Nonsteroidal anti‐inflammatory drugs reportedly reduce the risk of developing cancer. One mechanism by which they reduce carcinogenesis involves the inhibition of the activity of cyclooxygenase‐2, an enzyme that is overexpressed in various cancer tissues. Its overexpression increases cell proliferation and inhibits apoptosis. However, selected cyclooxygenase‐2 inhibitors can also act through cyclooxygenase‐independent mechanisms. In this study, using ultrafiltration, enzyme‐immobilized magnetic beads, high‐performance liquid chromatography, and electrospray‐ionization mass spectrometry, several isoflavonoids in Trifolium pratense L. extracts were screened and identified. Semi‐preparative high‐performance liquid chromatography and high‐speed counter‐current chromatography were then applied to separate the active constituents. Using these methods, seven major compounds were identified in Trifolium pratense L. As cyclooxygenase‐2 inhibitors: rothindin, ononin, daidzein, trifoside, pseudobaptigenin, formononetin, and biochanin A, which were then isolated with >92% purity. This is the first report of the presence of potent cyclooxygenase‐2 inhibitors in Trifolium pratense L. extracts. The results of this study demonstrate that the systematic isolation of bioactive components from Trifolium pratense L., by using ultrafiltration, enzyme‐immobilized magnetic beads, semi‐preparative high‐performance liquid chromatography, and high‐speed counter‐current chromatography, represents a feasible and efficient technique that could be extended for the identification and isolation of other enzyme inhibitors.     

Preparation of five high‐purity iridoid glycosides from Gardenia jasminoides Eills by molecularly imprinted solid‐phase extraction integrated with preparative liquid chromatography

Five iridoid glycosides were prepared using molecularly imprinted solid‐phase extraction combined with preparative high‐performance liquid chromatography. Hydrophilic molecularly imprinted polymers were synthesized using α‐1‐allyl‐2‐N‐acetyl glucosamine, which introduced an abundance of hydrophilic groups into the polymers. Using molecularly imprinted solid‐phase extraction as the sample pretreatment procedure, five iridoid glycosides, gardenoside, geniposide, shanzhiside, geniposidic acid, and genipin‐1‐O‐gentiobioside, were selectively enriched from Gardenia fructus extracts. Preparative high‐performance liquid chromatography then provided iridoid glycosides with a purity >98%. The structures were elucidated by using nuclear magnetic resonance spectroscopy, optical rotation and melting point measurements, and mass spectrometry. The results demonstrate that molecularly imprinted solid‐phase extraction combined with preparative high‐performance liquid chromatography was an efficient, rapid, and economical method for the preparation of bioactive compounds from natural products.     

Separation of α‐amylase inhibitors from Abelmoschus esculentus (L).Moench by on‐line two‐dimensional high‐speed counter‐current chromatography target‐guided by ultrafiltration‐HPLC

In this study, an on‐line two‐dimensional high‐speed counter‐current chromatography system based on a six‐port valve was developed. Target‐guided by ultrafiltration with high‐performance liquid chromatography, the one‐step isolation of three potential α‐amylase inhibitors from Abelmoschus esculentus (L).Moench was achieved by employing the developed orthogonal system and extrusion elution mode. The purities of three potential α‐amylase inhibitors were all over 95% as determined by high‐performance liquid chromatography. Furthermore, UV, mass spectrometry and ¹H NMR spectroscopy were applied to the structural identification of the isolated three target compounds, their structures were assigned as quercetin‐3‐O‐sophoroside (i), 5,7,3′,4′‐tetrahydroxy flavonol‐3‐O‐[β‐d ‐rhamnopyranosil‐(1→2)]‐β‐d ‐glucopyranoside (ii ) and isoquercitrin (iii), respectively. The Results demonstrated that the proposed method was highly efficient to screen and isolate enzyme inhibitors from complex natural products extracts, and on‐line two‐dimensional high‐speed counter‐current chromatography can effectively increase the peak resolution of target compounds.     

Ultrafiltration liquid chromatography combined with high‐speed countercurrent chromatography for screening and isolating potential α‐glucosidase and xanthine oxidase inhibitors from Cortex Phellodendri

Cortex Phellodendri is a typical Chinese herb with a large number of alkaloids existing in all parts of it. The most common methods for screening and isolating alkaloids are mostly labor intensive and time consuming. In this study, a new assay based upon ultrafiltration liquid chromatography was developed for the rapid screening of ligands for α‐glucosidase and xanthine oxidase. The C. Phellodendri extract was found to contain two alkaloids with both α‐glucosidase‐ and xanthine oxidase binding activities and one lactone with α‐glucosidase‐binding activity. Subsequently, with the help of high‐speed countercurrent chromatography, the specific binding ligands including palmatine, berberine, and obaculactone with purities of 97.38, 96.12, and 96.08%, respectively, were successfully separated. An optimized low‐toxicity two‐phase solvent system composed of ethyl acetate/n‐butanol/ethanol/water (3.5:1.7:0.5:5, v/v/v/v) was used to isolate the three compounds mentioned above from C. Phellodendri. The targeted compounds were identified by liquid chromatography coupled with mass spectrometry and NMR spectroscopy. Therefore, ultrafiltration liquid chromatography combined with high‐speed countercurrent chromatography is not only a powerful tool for screening and isolating α‐glucosidase and xanthine oxidase inhibitors in complex samples but is also a useful platform for discovering bioactive compounds for the prevention and treatment of diabetes mellitus and gout.     

Combinative application of pH‐zone‐refining and conventional high‐speed counter‐current chromatography for preparative separation of caged polyprenylated xanthones from gamboge

An efficient method for the preparative separation of four structurally similar caged xanthones from the crude extracts of gamboge was established, which involves the combination of pH‐zone‐refining counter‐current chromatography and conventional high‐speed counter‐current chromatography for the first time. pH‐zone‐refining counter‐current chromatography was performed with the solvent system composed of n‐hexane/ethyl acetate/methanol/water (7:3:8:2, v/v/v/v), where 0.1% trifluoroacetic acid was added to the upper organic stationary phase as a retainer and 0.03% triethylamine was added to the aqueous mobile phase as an eluter. From 3.157 g of the crude extract, 1.134 g of gambogic acid, 180.5 mg of gambogenic acid and 572.9 mg of a mixture of two other caged polyprenylated xanthones were obtained. The mixture was further separated by conventional high‐speed counter‐current chromatography with a solvent system composed of n‐hexane/ethyl acetate/methanol/water (5:5:10:5, v/v/v/v) and n‐hexane/methyl tert‐butyl ether/acetonitrile/water (8:2:6:4,v/v/v/v), yielding 11.6 mg of isogambogenic acid and 10.4 mg of β‐morellic acid from 218.0 mg of the mixture, respectively. The purities of all four of the compounds were over 95%, as determined by high‐performance liquid chromatography, and the chemical structures of the four compounds were confirmed by electrospray ionization mass spectrometry and NMR spectroscopy. The combinative application of pH‐zone‐refining counter‐current chromatography and conventional high‐speed counter‐current chromatography shows great advantages in isolating and enriching the caged polyprenylated xanthones.     

Separation and purification of 9,10‐dihydrophenanthrenes and bibenzyls from Pholidota chinensis by high‐speed countercurrent chromatography

Stilbenoids are the main components of leaves and stems of Pholidota chinensis. In the present investigation, high‐speed counter‐current chromatography was used for the separation and purification of two classes of stilbenoids, namely, bibenzyls and 9,10‐dihydrophenanthrenes, on a preparative scale from whole plants of P. chinensis with different solvent systems after silica gel column chromatography fractionation. n‐Hexane/ethyl acetate/methanol/water (1.2:1:1:0.8, v/v/v/v) was selected as the optimum solvent system to purify 1‐(3,4,5‐trimethoxyphenyl)‐1′,2′‐ethanediol ( 1 ), coelonin ( 2 ), 3,4′‐dihydroxy‐5,5′‐dimethoxybibenzyl ( 3 ), and 2,?7‐?dihydroxy‐?3,?4,?6‐?trimethoxy‐?9,?10‐?dihydrophenanthrene ( 4 ). While 2,7‐dihydroxy‐3,4,6‐trimethoxy‐?9,?10‐?dihydrophenanthrene ( 5 ), batatasin III ( 6 ), orchinol ( 7 ), and 3′‐O‐methylbatatasin III ( 8 ) were purified by n‐hexane/ethyl acetate/methanol/water (1.6:0.8:1.2:0.4, v/v/v/v). After the high‐speed counter‐current chromatography isolation procedure, the purity of all compounds was over 94% assayed by ultra high performance liquid chromatography. The chemical structure identification of all compounds was carried out by mass spectrometry and ¹H and ¹³C NMR spectroscopy. To the best of our knowledge, the current investigation is the first study for the separation and purification of bibenzyls and 9,10‐dihydrophenanthrenes by high‐speed counter‐current chromatography from natural resources.     

Screening and isolation of cyclooxygenase‐2 inhibitors from the stem bark of Phellodendron amurense Ruprecht by ultrafiltration with liquid chromatography and tandem mass spectrometry,and complex chromatography

Nonsteroidal anti‐inflammatory drugs appear to reduce the risk of developing cancer. One mechanism through which nonsteroidal anti‐inflammatory drugs act to prevent carcinogenesis is inhibition of the activity of the enzyme cyclooxygenase‐2. The cyclooxygenase‐2 inhibitors are widely used to reduce the risk of developing cancer. Natural products are considered to be a promising source of several novel cyclooxygenase‐2 inhibitors. Ultrafiltration with liquid chromatography and mass spectrometry is an efficient method that can be applied to rapidly screen and identify the ligands from the barks of Phellodendron amurense Ruprecht. A continuous online method comprised of pressurized liquid extraction, countercurrent chromatography, and semi‐preparative liquid chromatography was developed for the efficient scaled‐up production of eight compounds with high purities. The bioactivities of the separated compounds were assessed by an in vitro enzyme inhibition assay. The use of bioactivity screening method combined with preparation method of bioactive compounds and an in vitro enzyme inhibition assay facilitated the efficient screening and isolation of the cyclooxygenase‐2 inhibitors from complex samples. This could be used as an efficient method for the large‐scale production of functional ingredients.     

Application of high‐speed counter‐current chromatography and HPLC to separate and purify of three polyacetylenes from Platycodon grandiflorum

Three polyacetylenes were isolated and purified from Platycodon grandiflorum A. DC for the first time by high‐speed counter‐current chromatography using a two‐phase solvent system composed of hexane/ethyl acetate/methanol/water (1:31:1:31, v/v/v/v) and high‐performance liquid chromatography with an Agilent ZORBAX® SB‐C18 column (4.6 mm × 150 mm, 5 μm). After separation by high‐speed counter‐current chromatography and high‐performance liquid chromatography, we obtained 3.5 mg of platetyolin A, 4.1 mg of platetyolin B, and 18.1 mg of lobetyolin with purities of 97.2, 96.7, and 96.9%, respectively. The purity of each compound was assessed by high‐performance liquid chromatography and the chemical structures were evaluated by high‐resolution electrospray ionization time‐of‐flight mass spectrometry and one‐ and two‐dimensional NMR spectroscopy. Among the isolated compounds, platetyolin A and platetyolin B are newly reported compounds.     

Effect of inorganic salt on partition of high‐polarity parishins in two‐phase solvent systems and separation by high‐speed counter‐current chromatography from Gastrodia elata Blume

Parishins are high‐polarity and major bioactive constituents in Gastrodia elata Blume. In this study, the effect of several inorganic salts on the partition of parishins in two‐phase solvent systems was investigated. Adding ammonium sulfate, which has a higher solubility in water, was found to significantly promote the partition of parishins in the upper organic polar solvents. Based on the results, a two‐phase solvent system composed of butyl alcohol/acetonitrile/near‐saturated ammonium sulfate solution/water (1.5:0.5:1.2:1, v/v/v/v) was used for the purification of parishins by high‐speed counter‐current chromatography. Fractions obtained from high‐speed counter‐current chromatography were subjected to semi‐preparative high‐performance liquid chromatography to remove salt and impurities. As a result, parishin E (6.0 mg), parishin B (7.8 mg), parishin C (3.2 mg), gastrodin (15.3 mg), and parishin A (7.3 mg) were isolated from water extract of Gastrodia elata Blume (400 mg). These results demonstrated that adding inorganic salt that has high solubility in water to the two‐phase solvent system in high‐speed counter‐current chromatography was a suitable approach for the purification of high‐polarity compounds.     

First separation of four aromatic acids and two analogues with similar structures and polarities from Clematis akebioides by high‐speed counter‐current chromatography

In this paper, we report an efficient method by high‐speed counter‐current chromatography for the first separation of four aromatic acids and two analogs with similar structures and polarities from Clematis akebioides. First, the ethyl acetate extract was treated by silica gel column chromatography to enrich the target compounds. And then the fraction with target compounds were purified by high‐speed counter‐counter chromatography using a two‐phase solvent system consisting of chloroform/acetonitrile/water (10:6:4, v/v). The results showed high‐speed counter‐current chromatography could be a powerful technology for the separation of compounds with similar structures and polarities. Besides, it was found acetonitrile could be a good methanol substitute when a chloroform/methanol/water system could not provide a good separation factor. This study provides a reference for the separation of compounds from Clematis akebioides.     

Application of an efficient strategy based on liquid–liquid extraction,high‐speed counter‐current chromatography,and preparative HPLC for the rapid enrichment,separation, and purification of four anthraquinones from Rheum tanguticum

This study presents an efficient strategy based on liquid–liquid extraction, high‐speed counter‐current chromatography, and preparative HPLC for the rapid enrichment, separation, and purification of four anthraquinones from Rheum tanguticum. A new solvent system composed of petroleum ether/ethyl acetate/water (4:2:1, v/v/v) was developed for the liquid–liquid extraction of the crude extract from R. tanguticum. As a result, emodin, aloe‐emodin, physcion, and chrysophanol were greatly enriched in the organic layer. In addition, an efficient method was successfully established to separate and purify the above anthraquinones by high‐speed counter‐current chromatography and preparative HPLC. This study supplies a new alternative method for the rapid enrichment, separation, and purification of emodin, aloe‐emodin, physcione, and chrysophanol.     

Isolation and purification of macrocyclic components from Penicillium fermentation broth by high‐speed counter‐current chromatography

In this paper, high‐speed counter‐current chromatography (HSCCC), assisted with ESI‐MS, was first successfully applied to the preparative separation of three macrolide antibiotics, brefeldin A (12.6 mg, 99.0%), 7′‐O‐formylbrefeldin A (6.5 mg, 95.0%) and 7′‐O‐acetylbrefeldin A (5.0 mg, 92.3%) from the crude extract of the microbe Penicillium SHZK‐15. Considering the chemical nature and partition coefficient (K) values of the three target compounds, a two‐step HSCCC isolation protocol was developed in order to obtain products with high purity. In the two‐step method, the crude ethyl acetate extract was first fractionated and resulted in two peak fractions by HSCCC using solvent system n‐hexane/ethyl acetate/methanol/water (HEMWat) (3:7:5:5 v/v/v/v), then purified using solvent systems HEMWat (3:5:3:5 v/v/v/v) and HEMWat (7:3:5:5 v/v/v/v) for each fraction. The purities and structures of the isolated compounds were determined by HPLC, X‐ray crystallography, ESI‐MS and NMR. The results demonstrated that HSCCC is a fast and efficient technique for systematic isolation of bioactive compounds from the microbes.     

Effective on‐line high‐speed shear dispersing emulsifier technique coupled with high‐performance countercurrent chromatography method for simultaneous extraction and isolation of carotenoids from Lycium barbarum L. fruits

An efficient combination strategy based on high‐speed shear dispersing emulsifier technique and high‐performance countercurrent chromatography was developed for on‐line extraction and isolation of carotenoids from the fruits of Lycium barbarum. In this work, the high‐speed shear dispersing emulsifier technique has been employed to extract crude extracts using the upper phase of high‐performance countercurrent chromatography solvent system composed of n‐hexane?dichloromethane?acetonitrile (10:4:6.5, v/v) as the extraction solvent. At the separation stage, the high‐performance counter‐current chromatography process adopts elution–extrusion mode and the upper phase of the solvent system as stationary phase (reverse‐phase mode). As a result, three compounds including zeaxanthin, zeaxanthin monopalmitate, and zeaxanthin dipalmitate with purities of 89, 90, and 93% were successfully obtained in one extraction‐separation operation within 120 min. The targeted compounds were analyzed and identified by high‐performance liquid chromatography, mass spectrometry, and NMR spectroscopy. The results indicated that the present on‐line combination method could serve as a simple, rapid, and effective way to achieve weak polar and unstable compounds from natural products.     

Separation of betacyanins from flowers of Amaranthus cruentus L. in a polar solvent system by high‐speed counter‐current chromatography

Betacyanin extract of Amaranthus cruentus L. flowers was fractionated by semi‐preparative high‐speed counter‐current chromatography in a highly polar solvent system: propan‐1‐ol/acetonitrile/(NH₄)₂SO_{4satd. soln}/H₂O (1.0:0.5:1.2:1.0, v/v/v/v) in tail‐to‐head mode with 76% retention of the stationary phase. The crude extract as well as the fractions containing betacyanins were analyzed by liquid chromatography with tandem mass spectrometry as well as by high‐resolution ion‐trap time‐of‐flight mass spectrometry detection technique for the molecular formulae and multi‐step fragmentation pattern elucidation. Four betacyanins; namely, amaranthin, betanin, 6′‐O‐formyl‐amaranthin, and 6′‐O‐malonyl‐amaranthin as well as their diastereomeric forms differing in the configuration of the C‐15 carbon atom were identified in the fractions. Amaranthin was the dominant pigment in the extract and was additionally analyzed by nuclear magnetic resonance correlation techniques after the counter‐current chromatographic and high‐performance liquid chromatographic isolation. Betacyanins were highly enriched during a single high‐speed counter‐current chromatographic step; therefore, the tentative identification of new compounds for the whole Amaranthaceae family, 6′‐O‐formyl‐amaranthin and 6′‐O‐malonyl‐amaranthin was possible. Different elution profiles of the pigments observed in the counter‐current chromatographic system in comparison to high‐performance liquid chromatography system confirm a complementarity of both the techniques especially in the separation of diastereomeric pairs of betacyanins.     

Purification of flavonoids from licorice using an off‐line preparative two‐dimensional normal‐phase liquid chromatography/reversed‐phase liquid chromatography method

An orthogonal (71.9%) off‐line preparative two‐dimensional normal‐phase liquid chromatography/reversed‐phase liquid chromatography method coupled with effective sample pretreatment was developed for separation and purification of flavonoids from licorice. Most of the nonflavonoids were firstly removed using a self‐made Click TE‐Cys (60 μm) solid‐phase extraction. In the first dimension, an industrial grade preparative chromatography was employed to purify the crude flavonoids. Click TE‐Cys (10 μm) was selected as the stationary phase that provided an excellent separation with high reproducibility. Ethyl acetate/ethanol was selected as the mobile phase owing to their excellent solubility for flavonoids. Flavonoids co‐eluted in the first dimension were selected for further purification using reversed‐phase liquid chromatography. Multiple compounds could be isolated from one normal‐phase fraction and some compounds with bad resolution in one‐dimensional liquid chromatography could be prepared in this two‐dimensional system owing to the orthogonal separation. Moreover, this two‐dimensional liquid chromatography method was beneficial for the preparation of relatively trace flavonoid compounds, which were enriched in the first dimension and further purified in the second dimension. Totally, 24 flavonoid compounds with high purity were obtained. The results demonstrated that the off‐line two‐dimensional liquid chromatography method was effective for the preparative separation and purification of flavonoids from licorice.     

Ultrafiltration‐LC–MS combined with semi‐preparative HPLC for the simultaneous screening and isolation of lactate dehydrogenase inhibitors from Belamcanda chinensis

Stroke represents the fourth leading cause of death in the USA and the second leading cause of death worldwide. Lactate dehydrogenase inhibitors are widely used in the treatment of ischemic stroke and natural products are considered a promising source of novel lactate dehydrogenase inhibitors. In this study, we used PC12 cells to determine the protective effect of extracts from the herb Belamcanda chinensis following toxic challenge. Using ultrafiltration high‐performance liquid chromatography coupled with photo‐diode array detection and electrospray ionization mass spectrometry, we screened and identified isoflavonoids from Belamcanda chinensis extracts. Semi‐preparative high‐performance liquid chromatography was then applied to separate and isolate the active constituents. Using these methods, we identified six major compounds in Belamcanda chinensis as lactate dehydrogenase inhibitors: tectoridin, iristectorin A, iridin, tectorigenin, irigenin, and irisflorentin, which were then isolated to >92% purity. This is the first report that Belamcanda chinensis extracts contain potent lactate dehydrogenase inhibitors. Our results demonstrate that the systematic isolation of bioactive components from Belamcanda chinensis guided by ultrafiltration high‐performance liquid chromatography coupled with photo‐diode array detection and electrospray ionization mass spectrometry represents a feasible and efficient technique that could be extended for the identification and isolation of other enzyme inhibitors.     

Ionic‐liquid‐based ultrasound‐assisted extraction of isoflavones from Belamcanda chinensis and subsequent screening and isolation of potential α‐glucosidase inhibitors by ultrafiltration and semipreparative high‐performance liquid chromatography

The separation of a compound of interest from its structurally similar homologues to produce high‐purity natural products is a challenging problem. This work proposes a novel method for the separation of iristectorigenin A from its structurally similar homologues by ionic‐liquid‐based ultrasound‐assisted extraction and the subsequent screening and isolation of potential α‐glucosidase inhibitors via ultrafiltration and semipreparative high‐performance liquid chromatography. Ionic‐liquid‐based ultrasound‐assisted extraction was successfully applied to the extraction of tectorigenin, iristectorigenin A, irigenin, and irisflorentin from Belamcanda chinensis . The optimum conditions for the efficient extraction of isoflavones were determined as 1.0 M 1‐ethyl‐3‐methylimidazolium tetrafluoroborate with extraction time of 30 min and a solvent to solid ratio of 30 mL/g. Ultrafiltration with liquid chromatography and mass spectrometry was applied to screen and identify α‐glucosidase inhibitors from B. chinensis , followed by the application of semipreparative high‐performance liquid chromatography to separate and isolate the active constituents. Four major compounds including tectorigenin, iristectorigenin A, irigenin, and irisflorentin were screened and identified as α‐glucosidase inhibitors, and then the four active compounds abovementioned were subsequently isolated by semipreparative high‐performance liquid chromatography (99.89, 88.97, 99.79, and 99.97% purity, respectively). The results demonstrate that ionic liquid extraction can be successfully applied to the extraction of isoflavones from B. chinensis .     

Preparative separation of bioactive compounds from essential oil of Flaveria bidentis (L.) Kuntze using steam distillation extraction and one step high‐speed counter‐current chromatography

In order to utilize and control the invasive weed, bioactive compounds from essential oil of Flaveria bidentis (L.) Kuntze were studied. Steam distillation extraction and one step high‐speed counter‐current chromatography were applied to separate and purify the caryophyllene oxide, 7,11‐dimethyl‐3‐methylene‐1,6,10‐dodecatriene, and caryophyllene from essential oil of Flaveria bidentis (L.) Kuntze. The two‐phase solvent system containing n‐hexane/acetonitrile/ethanol (5:4:3, v/v/v) was selected for the one step separation mode according to the partition coefficient values (K) of the target compounds and the separation factor (α). The purity of each isolated fraction after a single high‐speed counter‐current chromatography run was determined by high performance liquid chromatography. A 3.2 mg of caryophyllene oxide at a purity of 92.6%, 10.4 mg of 7,11‐dimethyl‐3‐methylene‐1,6,10‐dodecatriene at a purity of 99.1% and 5.7 mg of caryophyllene at a purity of 98.8% were obtained from 200 mg essential oil of Flaveria bidentis (L.) Kuntze. The chemical structures of these components were identified by GC‐MS, ¹H‐NMR, and ¹³C‐NMR.     

Preparative separation of bioactive constitutes from Zanthoxylum planispinum using linear gradient counter‐current chromatography

Counter‐current chromatography is a chromatographic technique with a support‐free liquid stationary phase. In the present study, a successful application of linear gradient counter‐current chromatographic method for preparative isolation of bioactive components from the crude ethanol extract of Zanthoxylum planispinum was presented. The application of n‐hexane/ethyl acetate/methanol/water quaternary solvents, in terms of “HEMWat” or “Arizona” solvent families, in gradient elution mode was evaluated. Results indicated that slightly proportional changes of biphasic liquid systems provided the possibility of gradient elution in counter‐current chromatography, maintaining stationary phase retention in the column. With the selected quaternary solvent systems composed of n‐hexane/ethyl acetate/methanol/water (2:1:2:1 and 3:2:3:2, v/v), and optimized gradient programs, in total seven fractions were separated in 4.5 h. Most of the purified compounds could be obtained at the milligram level with over 80% purity. The present study indicated that the linear gradient counter‐current chromatographic approach possessed unique advantages in terms of separation efficiency, exhibiting great potential for the comprehensive separation of complex natural extracts.     

Development of a method to screen and isolate potential α‐glucosidase inhibitors from Panax japonicus C.A. Meyer by ultrafiltration,liquid chromatography,and counter‐current chromatography

A new assay based on ultrafiltration, liquid chromatography and mass spectrometry was developed for the rapid screening and identification of the ligands for α‐glucosidase from the extract of Panax japonicus. Six saponins were identified as α‐glucosidase inhibitors. Subsequently, the specific binding ligands, namely, notoginsenoside R₁, ginsenoside Rb₁, chikusetsusaponin V, chikusetsusaponin IV, chikusetsusaponin IVa, and ginsenoside Rd (the purities were 94.18, 95.43, 96.09, 93.26, 94.50, 93.86%, respectively) were separated by counter‐current chromatography using two‐phase solvent systems composed of tert‐butyl methyl ether, acetonitrile, 0.1% aqueous formic acid (3.8:1.0:4.4, v/v/v) and the solvent system composed of methylene chloride, isopropanol, methanol, 0.1% aqueous formic acid (5.8:1.0:6.0:2.2, v/v/v). The results demonstrate that ultrafiltration, liquid chromatography and mass spectrometry combined with high‐speed counter‐current chromatography might provide not only a powerful tool for screening and isolating α‐glucosidase inhibitors in complex samples but also a useful platform for discovering bioactive compounds for the prevention and treatment of diabetes mellitus.